Transmissible spongiform encephalopathies (TSEs) are a unique family of neurodegenerative diseases of the central nervous system that are always fatal. Of the various forms of prion disease, those with the most impact upon human and animal health are Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie and chronic wasting disease (CWD). The outbreaks of BSE and variant CJD (vCJD) in the United Kingdom and worldwide, including the United States, has prompted the need for rapid, reliable and inexpensive screening methods that detect TSE infection in humans and live animals. Current diagnostic tests for prion disease have, thus far, focused on detection of the causal agent of the disease, the abnormal prion protein, or individual proteins that correlate with the neurological disease. These tests are inadequate because they are post-mortem, low throughput and not sufficiently sensitive to detect infection early in the pre-clinical period. Clearly, there is an urgent need for the development of a reliable, sensitive, and specific ante-mortem diagnostic test for the pre-clinical identification of TSE-infected animals or individuals. Herein we propose the development of a new highly-sensitive mass spectrometry (MS)-based analytical platform in combination with a suite of bioinformatics tools to identify a panel of biomarkers of prion disease in body fluids collected from live animals infected with prion disease during the preclinical incubation period. The guiding hypotheses of the proposed research are: (1) The pathophysiological changes associated with prion infection will result in an altered protein profile of the cerebrospinal fluids (CSF) and/or serum proteome. (2) The combination of accurate mass measurements, isotopic labeling strategy for quantitative analyses, tandem mass spectrometry, and a suite of machine-learning algorithms will provide a unique and integrated platform to identify and characterize these diagnostic protein markers with unprecedented sensitivity and specificity. (3) Characterization of these diagnostic protein biomarkers will provide a basis for the development of an ante-mortem antibody-based screening test for prion diseases in suspected animals. To investigate these hypotheses, we propose the following specific aims: (1) To identify a panel of candidate biomarkers in the CSF and serum of animals infected with prion diseases using MS and classification algorithms. (2) To determine the identities of the candidate biomarkers by tandem MS and to obtain global quantitative changes of putative biomarkers via isotopic labeling. (3) To validate the biomarkers in blind diagnostic trials. Collectively, the proposed study will provide a basis for the development of a rapid and sensitive diagnosis of prion disease in an ante-mortem sample and it will also further our understanding of prion disease progression and pathology. This proposal explores a novel proteomic approach combining high sensitivity mass spectrometric profiling and sequencing with a set of bioinformatics tools to discover, identify, and quantify biomarkers that are indicative of prion infection. These putative biomarkers may also serve as targets for therapeutic intervention. Collectively, the proposed research will provide a basis for the development of a rapid and sensitive diagnosis of prion disease in an ante-mortem sample, enhancing our ability to secure food and blood supplies, and it will also further our understanding of prion disease progression and pathology.